The Role of Nitroreductase in the Reductive Metabolism of Flunitrazepam

• Main Authors: Yu-Lian Chen, 陳玉蓮
• Other Authors: 彭福佐
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/44728972618648457786

碩士 === 國立臺灣大學 === 毒理學研究所 === 94 === The benzodiazepine flunitrazepam is extensively prescribed to patients with insomnia in many countries, but has also become popular among drug abusers. In Taiwan, flunitrazepam (FNZ) has been prescribed by doctors for controlling hyponic, sedative, anxiolytic, muscle-relaxing and anticonvulsant effects of patients. FNZ has a nitro group and a fluorine atom in the molecule, both of them increase the hypnotic effect of benzodiazepine. In human, FNZ is oxidized to the major metabolites N-demethylflunitrazepam (NDF), 3-hydroxyflunitrazepam (3HF) and reduced to 7-aminoflunitrazepam (7-AF). It has been identified CYP2C19 and CYP3A4 as the principal cytochrome P450s involved in the metabolism of FNZ to its major metabolites N-DF and 3-HF. However, it is not clear which enzyme is responsible for the reduction of FNZ to 7-AF. The reduction of certain xenobiotics is also mediated by mixed and pure cultures of intestinal microflora. It therefore seemed worth investigating the role of intestinal microflora in the reduction of FNZ. The overarching goal of this study was to delineate the reductive metabolism of FNZ by intestine microflora. In addition, it has been reported that reduction of the nitro moiety to the corresponding amine is catalyzed by nitroreductase. Therefore, it led us to propose that nitroreductase is involved in the reductive metabolism of FNZ. Sex and age differences in the metabolism of xenobiotics are well known. It has been shown that nitroreductse expression was related to age and sex difference. Therefore, in this study, we also studied gender- and age-related changes in FNZ reductive metabolism in Wistar rats. In order to elucidate the role of nitroreductase in FNZ reductive metabolism, the experiments were performed with SKOV3NR cell line derived from human ovarian carcinoma cell line, in which E. coli. nitroreductase was expressed. Furthermore, the chemical inhibitor 4-nitrobenzoic acid (4-NBA) and 2-iodosobenzoic acid (2-IBA), E. coli. nitroreductase antibody and purified E. coli. nitroreductase enzyme were also used to identify the nitroreductase enzyme responsible for 7-AF formation. The following are the results obtained：(1) The amount of 7-AF formed from FNZ in intestinal microflora was higher than liver and intestine cytosol from 8-week-old Wistar rats. (2) The FNZ reductive metabolism was dose- and time- dependent in intestinal microflora from 8-week-old Wistar rats. (3) The amount of 7-AF formed from FNZ in 65-week-old Wistar rats was higher than in 1-week-old Wistar rats and also higher in female rats than in male rats. (4) 2-iodosobenzoic acid (2-IBA), a inhibitor of nitroreductase, significantly inhibited 7-AF formation. (5) The anti-nitroreductase anti-body also significantly inhibited 7-AF production. (6) Immunoblotting assay showed that nitroreductase protein was detected in liver, intestine and intestinal microflora cytosol of wistar rats. (7) Immunoblotting assay showed that nitroreductase protein was actually detected in SKOV3NR. (8) The FNZ reductive metabolism was dose- and time- dependent in SKOV3NR cell line and purified E. coli. nitroreductase. Therefore, these results suggested that intestinal nitroreductase plays an important role in the reductive metabolism pathway from FNZ to 7-AF.